The Vertebrate Embryology Section seeks to elucidate molecular and cellular events that drive gastrulation. During vertebrate gastrulation, embryos undergo dramatic morphogenesis, beginning as a simple assembly of homogenous cells, and emerging as highly diversified beings with a recognizable vertebrate body plan. Errors during gastrulation, due to genetic mutations or environmental insults, can lead to developmental deformities or miscarriage. A more precise understanding of gastrulation will enable the development of diagnostic and therapeutic tools for human developmental disorders. We have three major projects focused on elucidating mechanisms of gastrulation. Following is a summary of these projects and progress reports for Oct 1 2004-Sep 8 2005. Project 1. Characterization of the FoxH1 loss-of-function phenotype. FoxH1 is a co-transcription factor that acts in concert with Smad2 and 3. Injection of zebrafish embryos with antisense morpholino oligonucleotides (MOs) designed to block foxH1 translation severely perturbs early development. Our precise characterization of FoxH1 morphant embryos over the last 12 months has revealed that three key gastrulation movements are disrupted, and that a characteristic increase in the fraction of G1 phase cells is significantly delayed. Microarray analysis has also identified a number of downregulated targets, which we are currrently assessing. Project 2. Development and use of FAM-P. We have invented a new method for microdissection of embryos: FACS-Assisted Microdissection of Photoconverted cells (FAM-P). We are currently using FAM-P to profile regional differences in gene expression during the onset of gastrulation. Specifically we are contrasting gene expression in mesendoderm and neurectoderm precursor cells. Initial experiments have rapidly identified several undescribed mesendoderm-specific genes. Project 3. A genome-wide screen for essential RhoGEF genes. RhoGEFs are key positive regulators of RhoGTPases, a family of small G proteins that regulate various aspects of cellular morphogenesis via modulation of the actin cytoskeleton. To explore morphogenetic cascades that function during gastrulation, we have initiated a reverse-genetic screen to rapidly identify zebrafish RhoGEFs with essential functions during the first 24 hours of zebrafish development. During the past year we have demonstrated that 42 out of 71 predicted zebrafish RhoGEFs are transcribed during gastrulation. Loss-of-function analysis of 23 of these genes using injected antisense MOs designed to block splicing has already uncovered 6 with essential functions.